Although a variety of chemiluminescent and bioluminescent labels have been used to develop highly sensitive heterogeneous immunoassays, only a small number of homogeneous immunoassays based on these labels have been reported. This is a result of the properties of the labels themselves (i.e., quantum efficiency, stability, etc.) that preclude their use in the development of homogeneous assays with the desired sensitivity. In this study, we propose to develop and evaluate novel competitive binding assays for biomolecules by taking advantage of the selective interaction between biological binders (antibodies, binding proteins, receptors, etc.) and ligands, as well as the sensitivity associated with bioluminescence detection. The photoprotein aequorin will be used as the bioluminescent label. Different approaches to the synthesis of aequorin-ligand conjugates will be undertaken. Specifically, aequorin will be modified in a conventional way by attaching ligands to free amino groups on lysine residues of the protein. Further, mono-substituted aequorin-ligand conjugates (i.e., conjugates where one molecule of ligand is attached to specific locations on the protein molecule) will be prepared by genetic engineering of aequorin. We intend to study the effect that specific biological binders have on the luminescence activity of these aequorin- ligand conjugates. Moreover, systems in which the interaction between the conjugate and its corresponding biological binder causes Inhibition of the bioluminescence activity of the conjugate will be used to develop simple homogeneous assays for ligands in physiological samples. In cases where no substantial inhibition of the bioluminescence signal is observed, the development of solid-phase heterogeneous type assays will be investigated. In both cases, fundamental studies will be undertaken to investigate the nature of the observed assay characteristics and to improve the sensitivity and detection limits of the technique. It is anticipated that extremely sensitive assays for biologically important molecules will result from these investigations.
